| Gene name: | EGR2 |
| Aging type: | Accelerate |
| Aging characteristic: |
| Tissue type: | -- |
| Cell name: | DS HMFs |
| Experiment: | qRT-PCR//Immunofluorescence//ELISA |
| Description: | Using multi‐parameter phenotypic analysis to control for off‐target effects, we identified ‘EGR2 3’ siRNA as the most potent siRNA transfected individually which significantly increased cell number and significantly reversed cell area, nuclear area and cell elongation. ‘EGR2 2’, the second most potent siRNA transfected individually, produced a modest increase in cell number and significantly reversed nuclear area and cell elongation. Finally, the least potent siRNA, ‘EGR2 1’, only significantly reversed cell elongation compared to the DS?+?siGLO control. Further characterisation of the changes to the senescence phenotype following ablation of the?EGR2?in DS HMFs revealed a significant down‐regulation of the SASP factor,?IL‐6, at the transcript level and at the secreted protein level. |
| Target gene: | ARF//P16 |
| Official symbol(s): | ARF//P16 |
| R-AG-Target gene: | Upregulation |
| Subcategory: | Binding to promoter |
| Target gene experiment: | CHIP//Western blot |
| Target gene description: | Cells transfected with the pGL3 ARF 800 or with the complete ARF promoter, pGL3 ARF 3.4, displayed a significant increase in luciferase activity following transfection with the EGR2 expression vector or E2F1 positive control, but not EGR1, EGR3 or EGR4 expression vectors, thus confirming EGR2 as a direct activator of the ARF promoter.Validation of the interaction between EGR2 and the ARF promoter was performed using chromatin immunoprecipitation (ChIP) on cross‐linked DNA from quiescent interleukin‐2 (IL‐2)‐dependent Kit225 human T‐lymphocytes, with low levels of ARF expression and Kit225 cells following IL‐2 activation which results in increased ARF expression.Subsequently, ChIP was performed with polyclonal antibodies against EGR2 or E2F1, which acted as a positive control. Addition of IL‐2 to Kit225 cells resulted in significantly increased binding of E2F1 and EGR2 to the ARF promoter, demonstrating that EGR2 can be detected at the endogenous ARF promoter.Furthermore, ChIP performed in EP and DS HMFs revealed significantly increased binding of EGR2 to the p16 promoter in DS HMFs, thus confirming that EGR2 can bind to the endogenous p16 promoter |
| Regulatory pathway: | P16-PRB//ARF-P53-P21 |
| R-AG-Pathway: | Activation |
| Pathway experiment: | Immunofluorescence |
| Pathway description: | Using immunofluorescence staining and high‐content analysis, we further examined p16 and p21 on a cellular level and found a significant increase in the proportion of double negative (p16? p21?) ‘reversed’ cells in DS HMFs following EGR2 knockdown in combination with?p21?siRNA (‘DS?+?EGR2?+?p21?siRNA) compared to the DS?+?p21 siRNA HMFs, an increase similar to that seen in the reversed DS?+?p16?+?p21 HMFs. |
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